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Ligation Problem

Ligation Problem - Molecular Biology Techniques

Ligation Problem - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 03-08-2013, 04:30 PM
Pipette Filler
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Default Ligation Problem



I am having difficulty with a ligation, here are the details:

1. Vector: 13kb
2. Insert: 2.6Kb

Both vector and insert had to be partially digested with BstBI and gel purified because there were too many cut sites, leaving me with a someone small concentration of DNA. The 260/280 and 260/230 ratios were low so I did a subsequent ETOH precipitation and got concentrations of vector: 16.7ng/uL and insert: 8.2ng/uL, both in 15uL of ddH2O.

I treated my vector with antarctic phosphatase at 37degrees C for 1hr and inactivated it at 65 degrees C for 20m. The protocol suggested using 1ug of vector but I did not have that much so I used what I had.


I used T4 DNA ligase from invitrogen, 23degrees for 1hr. and then diluted with ddH2O 1:5
controls and results are as follows:
1. Cut vector and no insert with ligase - no colonies
2. uncut vector and no insert without ligase to check Ab resistance gene and competency of cells: many colonies

I transformed into brand new MAX efficiency DH5alpha cells from Invitrogen. 100uL of cells with 2uL of ligation reaction diluted 1:5. 30min on ice, 45s in 42degree water bath, 2 m on ice, added 900uL SOC medium,1hr 225RPM at 37 degrees C. Spread onto plates

I plated my ligation + SOC at 5 different concentrations, all brought to at least 100uL with SOC. 20uL being my lowest and 600uL (spun down and resuspended in 100uL SOC) was my highest.

Any help would be greatly appreciated!!! I tried this 2 more times before with subcloning efficiency cells and it didn't work then either.
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